Probe, probe set, probe-immobilized carrier, and genetic testing method

Abstract

A nucleic acid probe for classification of pathogenic bacterial species is capable of collectively detecting bacterial strains of the same species and differentially detecting them from other bacterial species. Any one of the base sequences of SEQ ID NOS. 92 to 93 and complementary or modified sequences thereof or a combination of at least two of them is used for detecting the gene of an infectious disease pathogenic bacterium.

1 . A probe for detecting a gene of infectious disease pathogenic bacterium,  Aeromonas hydrophila , having any one of the following base sequences (1) to (3):
 (1) GCCTAATACGTATCAACTGTGACGTTAC (SEQ ID NO. 92) or a complementary sequence thereof;   (2) GCCTAATACGTGTCAACTGTGACGTTAC (SEQ ID NO. 93) or a complementary sequence thereof; and   (3) a modified sequence prepared such that any one of the sequences of SEQ ID NOS. 92 to 93 and the complementary sequences thereof is subjected to base deletion, substitution, or addition as far as the modified sequence retains a function as the probe.   
     
     
         2 . A probe-immobilized carrier, wherein at least one first probe according to  claim 1  is arranged on a solid-phase carrier. 
     
     
         3 . A probe-immobilized carrier according to  claim 2 , wherein the probe-immobilized carrier comprises at least one second probe having any one of the base sequences of SEQ ID NOS. 35 to 94 as mentioned in the specification immobilized at a position spaced from the first probe. 
     
     
         4 . A kit for detecting a gene of  Aeromonas hydrophila , comprising:
 a probe according to  claim 1 ; and   a reagent for detecting a reaction between the probe and a target nucleic acid.   
     
     
         5 . A kit according to  claim 4 , wherein the reagent contains a primer for amplifying the gene of  Aeromonas hydrophila , and the primer includes:
 (2) an oligonucleotide having a base sequence of 5′ gcggcaggcctaacacatgcaag 3′ (SEQ ID NO: 2); and   (24) an oligonucleotide having a base sequence of 5′ atccagccgcaggttcccctac 3′ (SEQ ID NO: 24).   
     
     
         6 . A gene detection kit, comprising:
 a probe-immobilized carrier according to  claim 3 ; and   a reagent for detecting a reaction between the probes and a target nucleic acid,   wherein the reagent contains a primer including at least one oligonucleotide selected from the following items (1) to (21) and at least one oligonucleotide selected from the following items (22) to (28):   (1) an oligonucleotide having a base sequence of 5′ gcggcgtgcctaatacatgcaag 3′ (SEQ ID NO: 1);   (2) an oligonucleotide having a base sequence of 5′ gcggcaggcctaacacatgcaag 3′ (SEQ ID NO: 2);   (3) an oligonucleotide having a base sequence of 5′ gcggcaggcttaacacatgcaag 3′ (SEQ ID NO: 3);   (4) an oligonucleotide having a base sequence of 5′ gcggtaggcctaacacatgcaag 3′ (SEQ ID NO: 4);   (5) an oligonucleotide having a base sequence of 5′ gcggcgtgcttaacacatgcaag 3′ (SEQ ID NO: 5);   (6) an oligonucleotide having a base sequence of 5′ gcgggatgccttacacatgcaag 3′ (SEQ ID NO: 6);   (7) an oligonucleotide having a base sequence of 5′ gcggcatgccttacacatgcaag 3′ (SEQ ID NO: 7);   (8) an oligonucleotide having a base sequence of 5′ gcggcatgcttaacacatgcaag 3′ (SEQ ID NO: 8);   (9) an oligonucleotide having a base sequence of 5′ gcggcgtgcttaatacatgcaag 3′ (SEQ ID NO: 9);   (10) an oligonucleotide having a base sequence of 5′ gcggcaggcctaatacatgcaag 3′ (SEQ ID NO: 10);   (11) an oligonucleotide having a base sequence of 5′ gcgggatgctttacacatgcaag 3′ (SEQ ID NO: 11);   (12) an oligonucleotide having a base sequence of 5′ gcggcgtgcctaacacatgcaag 3′ (SEQ ID NO: 12);   (13) an oligonucleotide having a base sequence of 5′ gcggcgtgcataacacatgcaag 3′ (SEQ ID NO: 13);   (14) an oligonucleotide having a base sequence of 5′ gcggcatgcctaacacatgcaag 3′ (SEQ ID NO: 14);   (15) an oligonucleotide having a base sequence of 5′ gcggcgcgcctaacacatgcaag 3′ (SEQ ID NO: 15);   (16) an oligonucleotide having a base sequence of 5′ gcggcgcgcttaacacatgcaag 3′ (SEQ ID NO: 16);   (17) an oligonucleotide having a base sequence of 5′ gcgtcatgcctaacacatgcaag 3′ (SEQ ID NO: 17);   (18) an oligonucleotide having a base sequence of 5′ gcgataggcttaacacatgcaag 3′ (SEQ ID NO: 18);   (19) an oligonucleotide having a base sequence of 5′ gcgacaggcttaacacatgcaag 3′ (SEQ ID NO: 19);   (20) an oligonucleotide having a base sequence of 5′ gctacaggcttaacacatgcaag 3′ (SEQ ID NO: 20);   (21) an oligonucleotide having a base sequence of 5′ acagaatgcttaacacatgcaag 3′ (SEQ ID NO: 21);   (22) an oligonucleotide having a base sequence of 5′ atccagccgcaccttccgatac 3′ (SEQ ID NO: 22);   (23) an oligonucleotide having a base sequence of 5′ atccaaccgcaggttcccctac 3′ (SEQ ID NO: 23);   (24) an oligonucleotide having a base sequence of 5′ atccagccgcaggttcccctac 3′ (SEQ ID NO: 24);   (25) an oligonucleotide having a base sequence of 5′ atccagccgcaccttccggtac 3′ (SEQ ID NO: 25);   (26) an oligonucleotide having a base sequence of 5′ atccagcgccaggttcccctag 3′ (SEQ ID NO: 26);   (27) an oligonucleotide having a base sequence of 5′ atccagccgcaggttctcctac 3′ (SEQ ID NO: 27); and   (28) an oligonucleotide having a base sequence of 5′ atccagccgcacgttcccgtac 3′ (SEQ ID NO: 28).   
     
     
         7 . A probe set for detecting a gene of infectious disease pathogenic bacterium,  Aeromonas hydrophila , including at least two probes selected from the following items (A) to (H):
 (A) a probe having a base sequence represented by GCCTAATACGTATCAACTGTGACGTTAC (SEQ ID NO. 92);   (B) a probe having a base sequence represented by GCCTAATACGTGTCAACTGTGACGTTAC (SEQ ID NO. 93);   (C) a probe having a complementary sequence of the base sequence represented by SEQ ID NO. 92;   (D) a probe having a complementary sequence of the base sequence represented by SEQ ID NO. 93;   (E) a probe having a modified sequence obtained by base deletion, substitution, or addition on the base sequence represented by SEQ ID NO. 92 as far as it retains the function of a probe for detecting the gene of  Aeromonas hydrophila;      (F) a probe having a modified sequence obtained by base deletion, substitution, or addition on the base sequence represented by SEQ ID NO. 93 as far as it retains the function of a probe for detecting the gene of  Aeromonas hydrophila;      (G) a probe having a modified sequence obtained by base deletion, substitution, or addition on the complementary sequence of the base sequence represented by SEQ ID NO. 92 as far as it retains the function of a probe for detecting the gene of  Aeromonas hydrophila ; and   (H) a probe having a modified sequence obtained by base deletion, substitution, or addition on the complementary sequence of the base sequence represented by SEQ ID NO. 93 as far as it retains the function of a probe for detecting the gene of  Aeromonas hydrophila.      
     
     
         8 . A probe-immobilized carrier according to  claim 2 , wherein the at least one first probe includes at least two first probes selected from the following items (A) to (H):
 (A) a probe having a base sequence represented by GCCTAATACGTATCAACTGTGACGTTAC (SEQ ID NO. 92);   (B) a probe having a base sequence represented by GCCTAATACGTGTCAACTGTGACGTTAC (SEQ ID NO. 93);   (C) a probe having a complementary sequence of the base sequence represented by SEQ ID NO. 92;   (D) a probe having a complementary sequence of the base sequence represented by SEQ ID NO. 93;   (E) a probe having a modified sequence obtained by base deletion, substitution, or addition on the base sequence represented by SEQ ID NO. 92 as far as it retains the function of a probe for detecting the gene of  Aeromonas hydrophila;      (F) a probe having a modified sequence obtained by base deletion, substitution, or addition on the base sequence represented by SEQ ID NO. 93 as far as it retains the function of a probe for detecting the gene of  Aeromonas hydrophila;      (G) a probe having a modified sequence obtained by base deletion, substitution, or addition on the complementary sequence of the base sequence represented by SEQ ID NO. 92 as far as it retains the function of a probe for detecting the gene of  Aeromonas hydrophila ; and   (H) a probe having a modified sequence obtained by base deletion, substitution, or addition on the complementary sequence of the base sequence represented by SEQ ID NO. 93 as far as it retains the function of a probe for detecting the gene of  Aeromonas hydrophila , and wherein the at least two first probes are arranged on a solid-phase carrier at intervals from each other.   
     
     
         9 . A probe-immobilized carrier according to  claim 8 , wherein the probe-immobilized carrier comprises at least one second probe having any one of the base sequences of SEQ ID NOS. 35 to 94 as mentioned in the specification immobilized at a position spaced from the at least two first probes. 
     
     
         10 . A method of detecting a gene of  Aeromonas hydrophila  in an analyte by using a probe-immobilized carrier, comprising the steps of:
 (i) reacting the analyte with a probe-immobilized carrier according to  claim 2 ; and   (ii) detecting the presence or absence of a reaction of the probe on the probe-immobilized carrier with a nucleic acid in the analyte, or detecting the strength of a hybridization reaction of the probe on the probe-immobilized carrier with a nucleic acid in the analyte.   
     
     
         11 . A method according to  claim 10 , further comprising the step of carrying out PCR amplification of the target nucleic acid in the analyte by using a primer including the following oligonucleotides:
 (2) an oligonucleotide having a base sequence of 5′ gcggcaggcctaacacatgcaag 3′ (SEQ ID NO: 2); and   (24) an oligonucleotide having a base sequence of 5′ atccagccgcaggttcccctac 3′ (SEQ ID NO: 24).